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Suppression of nitric oxide production in activated murine peritoneal macrophages in vitro and ex vivo by Scrophularia striata ethanolic extract
Journal of Ethnopharmacology 124 (2009) 166–169
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Suppression of nitric oxide production in activated murine peritoneal macrophages in vitro and ex vivo by Scrophularia striata ethanolic extract Abbas Azadmehr a,b,∗ , Afshin Afshari b , Behzad Baradaran c , Reza Hajiaghaee a , Shamsali Rezazadeh a , Hamidreza Monsef-Esfahani d a
Pharmacognosy Department, Institute of Medicinal Plants, (ACECR), Tehran, Iran Department of Immunology, Faculty of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Pharmacognosy, Faculty of Pharmacy, Medical Sciences, University of Tehran, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 5 October 2008 Received in revised form 15 March 2009 Accepted 27 March 2009 Available online 5 April 2009 Keywords: Nitric oxide Scrophularia striata Peritoneal macrophages Inflammation
a b s t r a c t Ethnopharmacological relevance: Scrophularia striata (Scrophulariaceae), a traditional Iranian medicine, has been used for the treatment of allergy, rheumatics and chronic inflammatory disorders. Aim of the study: In the present study, we investigated the in vitro and ex vivo suppressive effects of Scrophularia striata ethanolic extract on nitric oxide production in mouse peritoneal macrophages. Materials and methods: Peritoneal macrophages were harvested by lavaging with ice cold phosphate buffer saline. Macrophages obtained from mice not treated were cultured with 10 g/mL lipopolysaccaride (LPS), 20 U/mL interferon-␥ (IFN-␥), and various concentrations of Scrophularia striata extract for the in vitro experiments and those obtained from mice treated with different doses of the extract for 7 days were cultured with 10 g/mL LPS, 20 U/mL IFN-␥ for the in vivo experiments. Nitrit levels were measured by using the diazotization method based on the Griess reaction, which is an indirect assay for NO production. Results: In vitro exposure of mouse peritoneal macrophages with various concentrations of Scrophularia striata extract (10, 50 and 100 g/mL) significantly suppressed NO production in a dose-dependent manner. In vivo administration of Scrophularia striata extract (50 and 100 mg/kg) to Balb/c mice inhibited LPS and IFN-␥ induced production of NO in the isolated mouse peritoneal macrophages ex vivo in a dose-dependent manner. Exposure to Scrophularia striata extract had no effect on cell viability. Conclusion: The results of the study demonstrated that the Scrophularia striata extract inhibit NO production in activated murine macrophages and we suggest that Scrophularia striata may be used in treating the inflammatory diseases. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In the search for anti-inflammatory natural products, an extract prepared from the aerial parts of Scrophularia striata Boiss (Scrophulariaceae) was selected. It was gathered from northeastern Iran, in the Ruin region. Many species of this genus have been used since ancient times as folk remedies to treat ailments such as scrophulas, scabies, tumors, eczema, psoriasis, inflammatory affections, etc. Some species in this genus have shown
Abbreviations: NO, nitric oxide; LPS, lipopolysaccaride; IFN-␥, interferon-␥; DMSO, dimethyl sulfoxide; PBS, phosphate buffer saline; NMMLA, NG -Monomethyll-arginine; OD, optical density; iNOS, inducible nitric oxide synthase; FBS, fetal bovine serum; S.D., standard deviation. ∗ Corresponding author at: Department of Immunology, Faculty of Medicine, Tarbiat Modares University, Al ahmad St, Tehran, Iran. Tel.: +98 261 4764021; fax: +98 261 4764021. E-mail address: [email protected] (A. Azadmehr). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.03.042
anti-inflammatory activity (Diaz et al., 2004; Bas et al., 2007a). However, no report on the effect of this species has been published in the literature. Nitric oxide (NO) is a short-lived biomolecule that mediates many biological functions, including host defense, vasoregulation, platelet aggregation and neurotransmission (Moncada et al., 1991; Nathan, 1992). The NO production by activated macrophages has been shown to mediate immune functions including antimicrobial and antitumor activities (MacMicking et al., 1997). However, excess NO production has been implicated in septic shock and autoimmune diseases (MacMicking et al., 1995; Guzik et al., 2003). Therefore, inhibition of NO production has become a therapeutic target of treatment for inflammatory diseases. There have been no previous studies of the effects of Scrophularia striata (S. striata) on NO production in mouse peritoneal macrophages. In the present study, we investigated the effects of S. striata ethanolic extract on NO production in activated murine macrophages.
A. Azadmehr et al. / Journal of Ethnopharmacology 124 (2009) 166–169
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2. Materials and methods 2.1. Plant collection The aerial parts of S. striata Boiss were collected from the northeastern part of Iran, in the Ruin region (1350 m above sea level) and were air dried at room temperature. A sample was authenticated by Dr. F. Attar, and a voucher specimen was preserved in the Faculty of Sciences’ Herbarium at Tehran University, Tehran, Iran (TUH No: 36501). 2.2. Total extract Aerial parts of the plant were dried, powdered (100 g) and macerated with an 80% ethanol solution for 3 days with three changes of the solution. The resulting extract was filtered and evaporated under vacuum into a dried powder extract (10.3 g, 10.3%). Plant extract was dissolved in dimethyl sulfoxide (DMSO) with the 0.1% (v/v) concentration that was not toxic on the macrophages and used at appropriate concentrations. 2.3. Experimental animals Male Balb/c mice (20–22 g) were obtained from Pasteur Institute, Tehran, Iran. Animal experiments were conducted in accordance with current ethical regulations on animal research. Mice were randomized and housed five to a cage in plastic cages. The animals were maintained under standard laboratory conditions of a temperature of 25 ± 2 ◦ C and a photoperiod of 12 h and received standard mouse chow and water ad libitum.
Fig. 1. In vitro effect of S. striata on LPS plus IFN-␥ induced NO production on mouse peritoneal macrophages. Peritoneal macrophages were incubated with 10 g/mL LPS plus 20 U/mL IFN-␥ and various concentrations of S. striata extract (1, 10, 50 and 100 g/mL) for 24 h. The nitrite content of culture media was analysed. Non-stimulated peritoneal macrophages was as negative control (C). NG -Monomethyl-l-arginine 1 mM was as NO inhibitor control (NMMLA). Column (vehicle) represents activated peritoneal macrophages with LPS plus IFN-␥ and the same dilution of dimethyl sulfoxide employed for extract in PBS. Data represent mean ± S.D. values from three independent experiments. *P < 0.05 compared with the LPS plus IFN-␥ treated cells (0 = positive control).
cells failing to exclude the dye were counted and expressed as a percentage of the total cells present.
2.4. In vivo exposure to S. striata 2.7. Nitric oxide assay For the in vivo study, each mouse in the groups containing five mice, was injected intraperitoneally (i.p.) with various concentrations of S. striata extract (10, 50 and 100 mg/kg) consecutively for 7 days. The control animals received the same amount of the vehicle control (e.g. the same dilution of dimethyl sulfoxide employed for extract in PBS). 2.5. Peritoneal macrophages isolation and cell culture For the in vitro and in vivo experiments peritoneal macrophages were harvested immediately by lavaging with ice cold sterile PBS. Cells were washed twice and plated in RPMI 1640 (Sigma Chemical Co.) medium containing 10% Fetal Bovine Serum (GIBCO), 100 U/mL penicillin/100 g/mL streptomycin (Sigma Chemical Co.) and incubated for 2 h at 37 ◦ C in 5% CO2 humidified incubator. After 2 h incubation, non-adherent cells were removed by gently washing with PBS and freshly prepared medium was added (Moretao et al., 2003). Briefly, the cells were cultured in 96-well plates with 200 l of culture medium until the cells reached confluence (approximately 200,000 cells per well). In order to stimulate inducible NO synthase, fresh cultures medium containing LPS (10 g/mL) plus IFN-␥ (20 U/ml) (R&D systems) and various concentrations (1, 10, 50 and 100 g/mL) of S. striata extract for the in vitro studies were added and cultured at 37 ◦ C in 5% CO2 humidified incubator for 24 h. NG -monomethyl-l-arginine (NMMLA, Sigma) was used as NO inhibitor control at different concentrations (0.1, 0.5 and 1 mM). 2.6. Cell viability Cell viability was checked by trypan blue exclusion test. An aliquot of the cell suspension was mixed with an equal volume of 0.4% (w/v) Trypan blue in PBS and incubated for 10 min. The
Nitrit levels were measured by using the diazotization method based on the Griess reaction, which is an indirect assay for NO production as described before (Green et al., 1982). Nitrit was measured by adding 100 l of Griess reagent (1% sulphanilamide and 0.1% naphthylenediamine in 5% phosphoric acid; Roche) to 100 l sample of the medium. The optical density at 540 nm (OD540) was measured with a microplate reader. The concentrations were calculated by comparison with OD540 of the standard solutions of sodium Nitrit prepared in culture medium. 2.8. Statistical analysis Data shown represent the mean and standard error. Statistical analyses were performed by one-way analysis of variance (ANOVA) to express the difference among the groups. All analyses were performed using SPSS software13. Data were considered statistically significant at P < 0.05. 3. Results 3.1. In vitro effect of S. striata on NO production by mouse peritoneal macrophages To evaluate the effect of S. striata extract on NO production of mouse peritoneal macrophages stimulated by LPS plus IFN-␥, peritoneal macrophages of Balb/c mice were collected and treated with 10 g/mL LPS plus 20 U/mL IFN-␥ and various concentrations of S. striata extract (1, 10, 50 and 100 g/mL) for 24 h. As shown in Fig. 1, S. striata significantly inhibited NO production (P < 0.05). No effect on the viability of peritoneal macrophages exposed to S. striata extract was observed.
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A. Azadmehr et al. / Journal of Ethnopharmacology 124 (2009) 166–169
Fig. 2. In vivo effect of S. striata (10, 50 and 100 mg/kg; i.p.) on NO production in mouse peritoneal macrophages. Groups of five mice each were injected i.p. 10, 50 and 100 mg/kg of S. striata extract consecutively for 7 days. The control animals received the same amount of the vehicle control (e.g. the same dilution of dimethyl sulfoxide employed for extract in PBS). On day 8 peritoneal macrophages were collected and incubated with 10 g/mL LPS plus 20 U/mL IFN-␥ for 24 h. The nitrite content of culture media was analysed. Data represent mean ± S.D. values from three independent experiments. *P < 0.05 compared with cells isolated from animals treated by the vehicle control.
3.2. Ex vivo effect of S. striata on NO production by mouse peritoneal macrophages To evaluate the potential of S. striata extract to modulate NO production ex vivo, male Balb/c mice were treated with the vehicle of the extract (e.g. the same dilution of dimethyl sulfoxide employed for extract in PBS) or increasing concentrations of extract (10, 50 and 100 mg/kg body weight) by i.p. injection for 7 days. On day 8 the animals were killed and peritoneal macrophages were collected and treated with 10 g/mL LPS plus 20 U/mL IFN-␥ for 24 h. As shown in Fig. 2, exposure to S. striata extract inhibited NO production stimulated by LPS plus IFN-␥ in a dose-dependent manner. Statistical significance was in the highest S. striata extract exposure group (50 and 100 mg/kg). No effect on the viability of peritoneal macrophages was observed using trypan blue exclusion cell viability was greater than 90% for all groups. 3.3. Blockage of NO production by addition of NG -monomethyl-l-arginine (NMMLA) as positive control NG -Monomethyl-l-arginine 1 mM used as NO inhibitor control. NO production by 10 g/mL LPS plus 20 U/mL IFN-␥ activated peritoneal macrophages was significantly inhibited (P < 0.001), when different concentrations (0.1, 0.5 and 1 mM) of NMMLA were applied. This inhibition showed a dose manner dependency (Fig. 3). 4. Discussion S. striata is a traditional herb in Iran and has been used to treat various inflammatory diseases such as allergy, rheumatics and chronic inflammatory disorders. Some species in this genus have shown anti-inflammatory activity (Schinella et al., 2002; Bas et al., 2007b). Previously, we demonstrated inhibitory effect of S. striata extract on matrix metalloproteinases (MMPs) (Hajiaghaee et al., 2007). However, no anti-inflammatory effect of this species has ever been reported. Therefore, in the present study, the effect of extracts of aerial parts of S. striata was examined on NO production in murine macrophages ex vivo at doses of 10, 50 and 100 mg/kg and in vitro at doses of 1, 10, 50 and 100 g/ml. NO is a short-lived free radical, synthesized from arginine, with extremely high reactivity and a variety of physiological activities involved in the regulation of blood vessel dilatation and immune response, and functions as a neurotrans-
Fig. 3. The effect of NO inhibitor control NG -Monomethyl-l-arginine (NMMLA) on activated peritoneal macrophages with 10 g/mL LPS plus 20 U/mL IFN-␥. First column (C) represents of non-activated peritoneal macrophages. Column (0) represents activated peritoneal macrophages with LPS plus IFN-␥ and without NMMLA (NO inhibitor). NO production by LPS plus IFN-␥ activated peritoneal macrophages was significantly (*P < 0.001) inhibited when different concentrations (0.1, 0.5 and 1 mM) of NMMLA were added. Data represent mean ± S.D. values from the three independent experiments.
mitter (Bredt and Snyder, 1994; Prast and Philippu, 2001; Ignarro, 2002). However, excess production of NO is associated with several diseases such as chronic inflammatory diseases, septic shock, and autoimmune diseases by increasing vascular permeability and the extravasations of fluid and proteins at the inflammatory sites (Kolb and Kolb-Bachofen, 1992). Therefore, inhibition of high-output NO production has been a therapeutic strategy for the treatment of various inflammatory diseases. In the present study, S. striata extract significantly inhibited NO production in mouse peritoneal resident macrophages activated by LPS plus IFN-␥ in a concentration dependent manner in vivo and in vitro. The suppressive effect was not due to cytotoxicity. As NO is a critical mediator in inflammation, increase NO production has been implicated in inflammatory diseases and autoimmunity. In conclusion, we have demonstrated that S. striata extract acts as a NO inhibitor in peritoneal macrophages. However, the effects of S. striata on NO production are not fully explained and this effect may be associated with one of the effective constituents of S. striata extract. Further identification of the mechanisms of NO inhibition seem to be required. Acknowledgements This study was supported by the Institute of Medicinal Plants of Iran (ACECR). This work was also partly supported by Faculty of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran. References Bas, E., Recio, M.C., Abdallah, M., Manez, S., Giner, R.M., Cerda-Nicolas, M., Rios, J.L., 2007a. Inhibition of the pro-inflammatory mediators production and anti-inflammatory effect of the iridoid scrovalentinoside. Journal of Ethnopharmacology 110, 419–427. Bas, E., Recio, M.C., Manez, S., Giner, R.M., Escandell, J.M., Lopez-Gines, C., Rios, J.L., 2007b. New insight into the inhibition of the inflammatory response to experimental delayed-type hypersensitivity reactions in mice by scropolioside A. European Journal of Pharmacology 555, 199–210. Bredt, D.S., Snyder, S.H., 1994. Nitric oxide: a physiologic messenger molecule. Annual Review of Biochemistry 63, 175–195. Diaz, A.M., Abad, M.J., Fernandez, L., Silvan, A.M., De Santos, J., Bermejo, P., 2004. Phenylpropanoid glycosides from Scrophularia scorodonia: in vitro antiinflammatory activity. Life Sciences 74, 2515–2526. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, Nitrit and [15 N] Nitrit in biological fluids. Analytical Biochemistry 126, 131–138.
A. Azadmehr et al. / Journal of Ethnopharmacology 124 (2009) 166–169 Guzik, T.J., Korbut, R., Adamek-Guzik, T., 2003. Nitric oxide and superoxide in inflammation and immune regulation. Journal of Physiology and Pharmacology 54, 469–487. Hajiaghaee, R., Monsef-Esfahani, H.R., Khorramizadeh, M.R., Saadat, F., Shahverdi, A.R., Attar, F., 2007. Inhibitory effect of aerial parts of Scrophularia striata on matrix metalloproteinases expression. Phytotherapy Research 21, 1127–1129. Ignarro, L.J., 2002. Nitric oxide in the regulation of vascular function: an historical overview. Journal of Cardiac Surgery 17, 301–306. Kolb, H., Kolb-Bachofen, V., 1992. Nitric oxide: a pathogenic factor in autoimmunity. Immunology Today 13, 157–160. MacMicking, J.D., Nathan, C., Hom, G., Chartrain, N., Fletcher, D.S., Trumbauer, M., Stevens, K., Xie, Q.W., Sokol, K., Hutchinson, N., 1995. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81, 641–650.
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MacMicking, J., Xie, Q.W., Nathan, C., 1997. Nitric oxide and macrophage function. Annual Review of Immunology 15, 323–350. Moncada, S., Palmer, R.M.J., Higgs, E.A., 1991. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacological Reviews 43, 109–142. Moretao, M.P., Buchi, D.F., Gorin, P.A.J., Iacomini, M., Oliveira, M.B.M., 2003. Effect of an acidic heteropolysaccharide (ARAGAL) from the gum of Anadenanthera colubrina (Angico branco) on peritoneal macrophage functions. Immunology Letters 89, 175–185. Nathan, C., 1992. Nitric oxide as a secretory product of mammalian cells. The Journal of the Federation of American Societies for Experimental Biology 6, 3051–3064. Prast, H., Philippu, A., 2001. Nitric oxide as a modulator of neuronal function. Progress in Neurobiology 64, 51–58. Schinella, G.R., Tournier, H.A., Prieto, J.M., Mordujovich de Buschiazzo, P., Rios, J.L., 2002. Antioxidant activity of anti-inflammatory plant extracts. Life Sciences 18, 1023–1033.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Suppression of nitric oxide production in activated murine peritoneal macrophages in vitro and ex vivo by Scrophularia striata ethanolic extract Abbas Azadmehr a,b,∗ , Afshin Afshari b , Behzad Baradaran c , Reza Hajiaghaee a , Shamsali Rezazadeh a , Hamidreza Monsef-Esfahani d a
Pharmacognosy Department, Institute of Medicinal Plants, (ACECR), Tehran, Iran Department of Immunology, Faculty of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Pharmacognosy, Faculty of Pharmacy, Medical Sciences, University of Tehran, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 5 October 2008 Received in revised form 15 March 2009 Accepted 27 March 2009 Available online 5 April 2009 Keywords: Nitric oxide Scrophularia striata Peritoneal macrophages Inflammation
a b s t r a c t Ethnopharmacological relevance: Scrophularia striata (Scrophulariaceae), a traditional Iranian medicine, has been used for the treatment of allergy, rheumatics and chronic inflammatory disorders. Aim of the study: In the present study, we investigated the in vitro and ex vivo suppressive effects of Scrophularia striata ethanolic extract on nitric oxide production in mouse peritoneal macrophages. Materials and methods: Peritoneal macrophages were harvested by lavaging with ice cold phosphate buffer saline. Macrophages obtained from mice not treated were cultured with 10 g/mL lipopolysaccaride (LPS), 20 U/mL interferon-␥ (IFN-␥), and various concentrations of Scrophularia striata extract for the in vitro experiments and those obtained from mice treated with different doses of the extract for 7 days were cultured with 10 g/mL LPS, 20 U/mL IFN-␥ for the in vivo experiments. Nitrit levels were measured by using the diazotization method based on the Griess reaction, which is an indirect assay for NO production. Results: In vitro exposure of mouse peritoneal macrophages with various concentrations of Scrophularia striata extract (10, 50 and 100 g/mL) significantly suppressed NO production in a dose-dependent manner. In vivo administration of Scrophularia striata extract (50 and 100 mg/kg) to Balb/c mice inhibited LPS and IFN-␥ induced production of NO in the isolated mouse peritoneal macrophages ex vivo in a dose-dependent manner. Exposure to Scrophularia striata extract had no effect on cell viability. Conclusion: The results of the study demonstrated that the Scrophularia striata extract inhibit NO production in activated murine macrophages and we suggest that Scrophularia striata may be used in treating the inflammatory diseases. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In the search for anti-inflammatory natural products, an extract prepared from the aerial parts of Scrophularia striata Boiss (Scrophulariaceae) was selected. It was gathered from northeastern Iran, in the Ruin region. Many species of this genus have been used since ancient times as folk remedies to treat ailments such as scrophulas, scabies, tumors, eczema, psoriasis, inflammatory affections, etc. Some species in this genus have shown
Abbreviations: NO, nitric oxide; LPS, lipopolysaccaride; IFN-␥, interferon-␥; DMSO, dimethyl sulfoxide; PBS, phosphate buffer saline; NMMLA, NG -Monomethyll-arginine; OD, optical density; iNOS, inducible nitric oxide synthase; FBS, fetal bovine serum; S.D., standard deviation. ∗ Corresponding author at: Department of Immunology, Faculty of Medicine, Tarbiat Modares University, Al ahmad St, Tehran, Iran. Tel.: +98 261 4764021; fax: +98 261 4764021. E-mail address: [email protected] (A. Azadmehr). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.03.042
anti-inflammatory activity (Diaz et al., 2004; Bas et al., 2007a). However, no report on the effect of this species has been published in the literature. Nitric oxide (NO) is a short-lived biomolecule that mediates many biological functions, including host defense, vasoregulation, platelet aggregation and neurotransmission (Moncada et al., 1991; Nathan, 1992). The NO production by activated macrophages has been shown to mediate immune functions including antimicrobial and antitumor activities (MacMicking et al., 1997). However, excess NO production has been implicated in septic shock and autoimmune diseases (MacMicking et al., 1995; Guzik et al., 2003). Therefore, inhibition of NO production has become a therapeutic target of treatment for inflammatory diseases. There have been no previous studies of the effects of Scrophularia striata (S. striata) on NO production in mouse peritoneal macrophages. In the present study, we investigated the effects of S. striata ethanolic extract on NO production in activated murine macrophages.
A. Azadmehr et al. / Journal of Ethnopharmacology 124 (2009) 166–169
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2. Materials and methods 2.1. Plant collection The aerial parts of S. striata Boiss were collected from the northeastern part of Iran, in the Ruin region (1350 m above sea level) and were air dried at room temperature. A sample was authenticated by Dr. F. Attar, and a voucher specimen was preserved in the Faculty of Sciences’ Herbarium at Tehran University, Tehran, Iran (TUH No: 36501). 2.2. Total extract Aerial parts of the plant were dried, powdered (100 g) and macerated with an 80% ethanol solution for 3 days with three changes of the solution. The resulting extract was filtered and evaporated under vacuum into a dried powder extract (10.3 g, 10.3%). Plant extract was dissolved in dimethyl sulfoxide (DMSO) with the 0.1% (v/v) concentration that was not toxic on the macrophages and used at appropriate concentrations. 2.3. Experimental animals Male Balb/c mice (20–22 g) were obtained from Pasteur Institute, Tehran, Iran. Animal experiments were conducted in accordance with current ethical regulations on animal research. Mice were randomized and housed five to a cage in plastic cages. The animals were maintained under standard laboratory conditions of a temperature of 25 ± 2 ◦ C and a photoperiod of 12 h and received standard mouse chow and water ad libitum.
Fig. 1. In vitro effect of S. striata on LPS plus IFN-␥ induced NO production on mouse peritoneal macrophages. Peritoneal macrophages were incubated with 10 g/mL LPS plus 20 U/mL IFN-␥ and various concentrations of S. striata extract (1, 10, 50 and 100 g/mL) for 24 h. The nitrite content of culture media was analysed. Non-stimulated peritoneal macrophages was as negative control (C). NG -Monomethyl-l-arginine 1 mM was as NO inhibitor control (NMMLA). Column (vehicle) represents activated peritoneal macrophages with LPS plus IFN-␥ and the same dilution of dimethyl sulfoxide employed for extract in PBS. Data represent mean ± S.D. values from three independent experiments. *P < 0.05 compared with the LPS plus IFN-␥ treated cells (0 = positive control).
cells failing to exclude the dye were counted and expressed as a percentage of the total cells present.
2.4. In vivo exposure to S. striata 2.7. Nitric oxide assay For the in vivo study, each mouse in the groups containing five mice, was injected intraperitoneally (i.p.) with various concentrations of S. striata extract (10, 50 and 100 mg/kg) consecutively for 7 days. The control animals received the same amount of the vehicle control (e.g. the same dilution of dimethyl sulfoxide employed for extract in PBS). 2.5. Peritoneal macrophages isolation and cell culture For the in vitro and in vivo experiments peritoneal macrophages were harvested immediately by lavaging with ice cold sterile PBS. Cells were washed twice and plated in RPMI 1640 (Sigma Chemical Co.) medium containing 10% Fetal Bovine Serum (GIBCO), 100 U/mL penicillin/100 g/mL streptomycin (Sigma Chemical Co.) and incubated for 2 h at 37 ◦ C in 5% CO2 humidified incubator. After 2 h incubation, non-adherent cells were removed by gently washing with PBS and freshly prepared medium was added (Moretao et al., 2003). Briefly, the cells were cultured in 96-well plates with 200 l of culture medium until the cells reached confluence (approximately 200,000 cells per well). In order to stimulate inducible NO synthase, fresh cultures medium containing LPS (10 g/mL) plus IFN-␥ (20 U/ml) (R&D systems) and various concentrations (1, 10, 50 and 100 g/mL) of S. striata extract for the in vitro studies were added and cultured at 37 ◦ C in 5% CO2 humidified incubator for 24 h. NG -monomethyl-l-arginine (NMMLA, Sigma) was used as NO inhibitor control at different concentrations (0.1, 0.5 and 1 mM). 2.6. Cell viability Cell viability was checked by trypan blue exclusion test. An aliquot of the cell suspension was mixed with an equal volume of 0.4% (w/v) Trypan blue in PBS and incubated for 10 min. The
Nitrit levels were measured by using the diazotization method based on the Griess reaction, which is an indirect assay for NO production as described before (Green et al., 1982). Nitrit was measured by adding 100 l of Griess reagent (1% sulphanilamide and 0.1% naphthylenediamine in 5% phosphoric acid; Roche) to 100 l sample of the medium. The optical density at 540 nm (OD540) was measured with a microplate reader. The concentrations were calculated by comparison with OD540 of the standard solutions of sodium Nitrit prepared in culture medium. 2.8. Statistical analysis Data shown represent the mean and standard error. Statistical analyses were performed by one-way analysis of variance (ANOVA) to express the difference among the groups. All analyses were performed using SPSS software13. Data were considered statistically significant at P < 0.05. 3. Results 3.1. In vitro effect of S. striata on NO production by mouse peritoneal macrophages To evaluate the effect of S. striata extract on NO production of mouse peritoneal macrophages stimulated by LPS plus IFN-␥, peritoneal macrophages of Balb/c mice were collected and treated with 10 g/mL LPS plus 20 U/mL IFN-␥ and various concentrations of S. striata extract (1, 10, 50 and 100 g/mL) for 24 h. As shown in Fig. 1, S. striata significantly inhibited NO production (P < 0.05). No effect on the viability of peritoneal macrophages exposed to S. striata extract was observed.
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A. Azadmehr et al. / Journal of Ethnopharmacology 124 (2009) 166–169
Fig. 2. In vivo effect of S. striata (10, 50 and 100 mg/kg; i.p.) on NO production in mouse peritoneal macrophages. Groups of five mice each were injected i.p. 10, 50 and 100 mg/kg of S. striata extract consecutively for 7 days. The control animals received the same amount of the vehicle control (e.g. the same dilution of dimethyl sulfoxide employed for extract in PBS). On day 8 peritoneal macrophages were collected and incubated with 10 g/mL LPS plus 20 U/mL IFN-␥ for 24 h. The nitrite content of culture media was analysed. Data represent mean ± S.D. values from three independent experiments. *P < 0.05 compared with cells isolated from animals treated by the vehicle control.
3.2. Ex vivo effect of S. striata on NO production by mouse peritoneal macrophages To evaluate the potential of S. striata extract to modulate NO production ex vivo, male Balb/c mice were treated with the vehicle of the extract (e.g. the same dilution of dimethyl sulfoxide employed for extract in PBS) or increasing concentrations of extract (10, 50 and 100 mg/kg body weight) by i.p. injection for 7 days. On day 8 the animals were killed and peritoneal macrophages were collected and treated with 10 g/mL LPS plus 20 U/mL IFN-␥ for 24 h. As shown in Fig. 2, exposure to S. striata extract inhibited NO production stimulated by LPS plus IFN-␥ in a dose-dependent manner. Statistical significance was in the highest S. striata extract exposure group (50 and 100 mg/kg). No effect on the viability of peritoneal macrophages was observed using trypan blue exclusion cell viability was greater than 90% for all groups. 3.3. Blockage of NO production by addition of NG -monomethyl-l-arginine (NMMLA) as positive control NG -Monomethyl-l-arginine 1 mM used as NO inhibitor control. NO production by 10 g/mL LPS plus 20 U/mL IFN-␥ activated peritoneal macrophages was significantly inhibited (P < 0.001), when different concentrations (0.1, 0.5 and 1 mM) of NMMLA were applied. This inhibition showed a dose manner dependency (Fig. 3). 4. Discussion S. striata is a traditional herb in Iran and has been used to treat various inflammatory diseases such as allergy, rheumatics and chronic inflammatory disorders. Some species in this genus have shown anti-inflammatory activity (Schinella et al., 2002; Bas et al., 2007b). Previously, we demonstrated inhibitory effect of S. striata extract on matrix metalloproteinases (MMPs) (Hajiaghaee et al., 2007). However, no anti-inflammatory effect of this species has ever been reported. Therefore, in the present study, the effect of extracts of aerial parts of S. striata was examined on NO production in murine macrophages ex vivo at doses of 10, 50 and 100 mg/kg and in vitro at doses of 1, 10, 50 and 100 g/ml. NO is a short-lived free radical, synthesized from arginine, with extremely high reactivity and a variety of physiological activities involved in the regulation of blood vessel dilatation and immune response, and functions as a neurotrans-
Fig. 3. The effect of NO inhibitor control NG -Monomethyl-l-arginine (NMMLA) on activated peritoneal macrophages with 10 g/mL LPS plus 20 U/mL IFN-␥. First column (C) represents of non-activated peritoneal macrophages. Column (0) represents activated peritoneal macrophages with LPS plus IFN-␥ and without NMMLA (NO inhibitor). NO production by LPS plus IFN-␥ activated peritoneal macrophages was significantly (*P < 0.001) inhibited when different concentrations (0.1, 0.5 and 1 mM) of NMMLA were added. Data represent mean ± S.D. values from the three independent experiments.
mitter (Bredt and Snyder, 1994; Prast and Philippu, 2001; Ignarro, 2002). However, excess production of NO is associated with several diseases such as chronic inflammatory diseases, septic shock, and autoimmune diseases by increasing vascular permeability and the extravasations of fluid and proteins at the inflammatory sites (Kolb and Kolb-Bachofen, 1992). Therefore, inhibition of high-output NO production has been a therapeutic strategy for the treatment of various inflammatory diseases. In the present study, S. striata extract significantly inhibited NO production in mouse peritoneal resident macrophages activated by LPS plus IFN-␥ in a concentration dependent manner in vivo and in vitro. The suppressive effect was not due to cytotoxicity. As NO is a critical mediator in inflammation, increase NO production has been implicated in inflammatory diseases and autoimmunity. In conclusion, we have demonstrated that S. striata extract acts as a NO inhibitor in peritoneal macrophages. However, the effects of S. striata on NO production are not fully explained and this effect may be associated with one of the effective constituents of S. striata extract. Further identification of the mechanisms of NO inhibition seem to be required. Acknowledgements This study was supported by the Institute of Medicinal Plants of Iran (ACECR). This work was also partly supported by Faculty of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran. References Bas, E., Recio, M.C., Abdallah, M., Manez, S., Giner, R.M., Cerda-Nicolas, M., Rios, J.L., 2007a. Inhibition of the pro-inflammatory mediators production and anti-inflammatory effect of the iridoid scrovalentinoside. Journal of Ethnopharmacology 110, 419–427. Bas, E., Recio, M.C., Manez, S., Giner, R.M., Escandell, J.M., Lopez-Gines, C., Rios, J.L., 2007b. New insight into the inhibition of the inflammatory response to experimental delayed-type hypersensitivity reactions in mice by scropolioside A. European Journal of Pharmacology 555, 199–210. Bredt, D.S., Snyder, S.H., 1994. Nitric oxide: a physiologic messenger molecule. Annual Review of Biochemistry 63, 175–195. Diaz, A.M., Abad, M.J., Fernandez, L., Silvan, A.M., De Santos, J., Bermejo, P., 2004. Phenylpropanoid glycosides from Scrophularia scorodonia: in vitro antiinflammatory activity. Life Sciences 74, 2515–2526. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, Nitrit and [15 N] Nitrit in biological fluids. Analytical Biochemistry 126, 131–138.
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